Determination of nitrogen mustard metabolites in urine using high-performance liquid chromatography and high-resolution mass spectrometry

Detection of biologically active components in mixtures of complex composition which include biological fluids (blood, urine, etc.) is the most difficult problem of the analytical chemistry and requires the use of modern highly informative research methods. One of the most effective and universal ways to study the structure of unknown substances is the method of liquid chromatography- high resolution mass spectrometry that combines the possibility of highly selective separation of the mixtures under study, the reliable detection of unknown substances and high sensitivity of the procedure. We propose a method for the simultaneous extraction of highly polar biomarkers of nitrogen mustard — N-triethanolamine (TEA), N-ethyl diethanolamine (EDEA) and N-methyldiethanolamine (MDEA) — from urine with subsequent determination by HPLC and detection by high resolution tandem mass spectrometry. The mass spectra of fragmentation of protonated molecular ions of TEA, EDEA, and MDEA have been studied and possible structural formulas of the fragment ions are given. The sample preparation of urine and mass spectrometric detection in the multiple reaction monitoring mode were optimized. The five-fold dilution with deionized water was chosen as a method of urine sample preparation for analysis. Separation of the components was performed in the reversed-phase chromatography mode with retention times for TEA, EDEA, and MDEA of 2.00, 2.05, and 1.92 min, respectively. The time required to complete all steps of the urine sample analysis did not exceed 25 min. The detection limits in urine were 1 ng/ml for TEA and 2 ng/ml for EDEA and MDEA. The developed approach provides determination of the fact of application of specific nitrogen mustard in enquiry of possible exposure of living organism to the blister agents.